Dynamics and Energy Contributions for Transport of Unfolded Pertactin through a Protein Nanopore

Abstract

To evaluate the physical parameters governing translocation of an unfolded protein across a lipid bilayer, we studied protein transport through aerolysin, a passive protein channel, at the single molecule level. The protein model used was the passenger domain of pertactin, an autotransporter virulence protein. Transport of pertactin through the aerolysin nanopore was detected as transient partial current blockades as the unfolded protein partially occluded the aerolysin channel. We compared the dynamics of entry and transport for unfolded pertactin and a covalent end-to-end dimer of the same protein. For both the monomer and the dimer, the event frequency of current blockades increased exponentially with the applied voltage, while the duration of each event decreased exponentially as a function of the electrical potential. The blockade time was twice as long for the dimer as for the monomer. The calculated activation free energy includes a main enthalpic component that we attribute to electrostatic interactions between pertactin and the aerolysin nanopore (despite the low Debye length), plus an entropic component due to confinement of the unfolded chain within the narrow pore. Comparing our experimental results to previous studies and theory suggests that unfolded proteins cross the membrane by passing through the nanopore in a somewhat compact conformation according to the “blob” model of Daoud and de Gennes.

Single-Molecule Dynamics and Discrimination between Hydrophilic and Hydrophobic Amino Acids in Peptides, through Controllable, Stepwise Translocation across Nanopores<strong> </strong>

“…4, panel Boltzmann constant, Tm the absolute temperature and I= ∑ is the ionic strenght of electrolyte, zi and Ci stand for the counterions valence number and their concentration in molar units), which is low relative to the average diameter of the α-HL's β-barrel (∼20 Å) or is vestibule (∼46 Å). For our system, the average diameters of the peptide's ends estimated with the Swiss-PdbViewer were ∼13.2 Å (E12) and ∼18 Å (R12); knowing that the diameter of the α-HL's β-barrel is ∼20 Å, it is conceivable that the peptide's ends will still experience electrostatic interactions with the inner surface of the β-barrel, in agreement with previous findings on different systems [36,59].…”

Nanopore Sensing

“…Later, a systematic study was undertaken by Clark and co-workers to obtain more in-depth information about the translocation dynamics of unfolded protein molecules. 145 They found that the translocation of unfolded protein molecules is mainly an enthalpically driven process due to the electrostatic interaction between the protein molecules and the pore, with a minor entropy component from the confinement of the proteins within the pore.…”

The Effects of Tetramethylammonium Cation on Oligonucleotide Analysis with Aerolysin Nanopore

“…[18] In 2006, Aerolysin was firstly applied to analyze the structure of α-helix peptides as a nanopore sensor. [19] Subsequently, it was used to detect various molecules, including oligosaccharides, [20] peptides, [21] proteins [22][23][24] and poly (ethylene glycols) (PEGs). [25] Since the diameter of aerolysin nanopore precisely allows for the passage of single-stranded DNA, aerolysin nanopore was demonstrated that could discriminate oligonucleotides of different lengths directly and real-time monitor the stepwise cleavage of Poly(dA) 5 by Exonuclease I.…”